Prion diseases, or transmissible spongiform encephalopathies, are fatal neurodegenerative conditions of humans and animals that have significantly impacted public health and the safety of the food and blood supplies. These disorders are caused by infectious proteins called prions, which propagate themselves by a self-templating mechanism in which PrPSc, the infectious isoform, seeds conformational conversion of PrPC, a normal, neuronal glycoprotein, into additional molecules of PrPSc. Although the mechanism by which infectious prions propagate is now well understood, the process by which they actually cause neuronal damage has remained mysterious. Most previous studies have been performed with proteins, cells, and tissues derived from mice and therefore they are limited in their ability to probe the genetic and cellular factors that contribute specifically to the pathogenesis of human prion diseases. Moreover, therapeutic agents developed using mouse models are often ineffective when tested in humans. As a result, there are currently no effective therapies or even palliative treatments for prion disorders. The overall goals of this proposal are to establish a powerful, new experimental platform, based on human induced pluripotent stem cells (iPSCs) and their differentiated neural progeny, to study the mechanisms by which prions damage neurons, and to test the efficacy of novel therapeutic compounds in a system that is directly relevant to human disease. The capacity of iPSCs to be expanded indefinitely in vitro, together with their ability to differentiate into any desired cell lineage (including neurons) provides a virtually inexhaustible source of patient-specific cells in which to analyze the direct effects of any disease-associated protein. In this proposal, we will utilize cultures of iPSC-derived cortical neurons from patients carrying the highly penetrant E200K PrP mutation associated with an inherited form of Creutzfeldt-Jakob disease. As a unique resource, we have access to samples from many members of a large Israeli family carrying this mutation, a number of which we have already converted into iPSC lines. Our specific aims include: (1) Characterizing human iPSC-derived neurons expressing E200K PrP in terms of their cellular, biochemical, and transcriptional profiles; and (2) Determining how expression of E200K PrP in iPSC-derived neurons affects synaptic morphology and function, and testing the effects of selected therapeutic compounds known to rescue synaptic defects. Because of the pathogenic commonalities between prion diseases and other protein aggregation disorders of the CNS, including Alzheimer's disease, we anticipate that the results obtained here will have wide applicability to a range of human neurodegenerative conditions.